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Method for separating and purifying exenatide on large scale

A technology for separation and purification of exenatide, which is applied in the field of large-scale separation and purification of polypeptides, can solve the problems of short production cycle, long production cycle, and high cost of separation and purification of exenatide, so as to save costs, increase product output and Yield effect

Active Publication Date: 2011-10-19
滨海吉尔多肽有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for large-scale separation and purification of exenatide. Its technical idea is to use a large-scale dynamic axial compression column to separate and purify exenatide on a large scale according to reversed-phase high-performance liquid chromatography, and to use a large-scale The dynamic axial compression column converts it into the form of acetate to achieve short production cycle, high efficiency, low cost and large-scale, mainly solving the problems of high cost, low yield and long production cycle in the existing separation and purification of exenatide technical problem

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Sample treatment: Weigh 80-100g of crude exenatide and dissolve it in 10L of acetic acid-acetonitrile aqueous solution with a volume ratio: acetic acid: acetonitrile: water = 1:5:40, ultrasonically treat the sample until it is completely clarified, and use a pore size of 0.45 Filter through a μm filter, and collect the filtrate for later use.

[0023] 2. Purification:

[0024] Purification conditions: Chromatographic column: DAC-HB200 dynamic axial compression column with octadecylsilane bonded silica gel as the stationary phase, column diameter and packing length: 20cm×28cm. Mobile phase: A: 0.1% acetic acid aqueous solution by mass percent; B: 0.1% acetic acid acetonitrile solution by mass percent. Flow rate: 1000ml / min. Detection wavelength: 230nm. Gradient: 0 to 40 minutes A:B from 90:10 to 10:90. The loading amount is 80g.

[0025] Purification process: wash the dynamic axial compression column with 0.1% acetic acid acetonitrile solution accounting for 80% ...

Embodiment 2

[0028] 1. Sample treatment: Weigh 80-100g of crude exenatide and dissolve it in 10L of acetic acid-acetonitrile aqueous solution with a volume ratio: acetic acid: acetonitrile: water = 1:5:40, ultrasonically treat the sample until it is completely clarified, and use a pore size of 0.45 Filter through a μm filter, and collect the filtrate for later use.

[0029] 2. Purification:

[0030] Purification conditions: Chromatographic column: DAC-HB200 dynamic axial compression column with octadecylsilane bonded silica gel as the stationary phase, column diameter and packing length: 20cm×28cm. Mobile phase: A: 0.2% acetic acid aqueous solution by mass percentage; B: 0.2% acetic acid acetonitrile solution by mass percentage. Flow rate: 1000ml / min. Detection wavelength: 230nm. Gradient: 0 to 40 minutes A:B from 80:20 to 20:80. The loading amount is 90g.

[0031] Purification process: Rinse the dynamic axial compression column with 0.2% acetic acid acetonitrile solution accounting f...

Embodiment 3

[0034] 1. Sample treatment: Weigh 80-100g of crude exenatide and dissolve it in 10L of acetic acid-acetonitrile aqueous solution with a volume ratio: acetic acid: acetonitrile: water = 1:5:40, ultrasonically treat the sample until it is completely clarified, and use a pore size of 0.45 Filter through a μm filter, and collect the filtrate for later use.

[0035] 2. Purification:

[0036] Purification conditions: Chromatographic column: DAC-HB200 dynamic axial compression column with octadecylsilane bonded silica gel as the stationary phase, column diameter and packing length: 20cm×28cm. Mobile phase: A: 0.3% acetic acid aqueous solution by mass percent; B: 0.3% acetic acid acetonitrile solution by mass percent. Flow rate: 1000ml / min. Detection wavelength: 230nm. Gradient: 0 to 40 minutes A:B from 70:30 to 30:70. The loading amount is 100g.

[0037] Purification process: Rinse the dynamic axial compression column with 0.3% acetic acid acetonitrile solution accounting for 80% ...

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Abstract

The invention relates to a method for separating and purifying exenatide on a large scale and in particular relates to a method for separating and purifying exenatide by utilizing a reversed-phase high performance liquid chromatography (RP-HPLC) on a large scale, and the method can be used for solving the problems of high cost, low yield, long production period and the like caused by utilizing the RP-HPLC to separate and purify the exenatide. The method comprises the following steps: 1) dissolving crude exenatide in an acetic acid-acetonitrile aqueous solution, carrying out ultrasonic processing and membrane filtration, and then taking out filtrate for later use; 2) separating and purifying a filtrated crude product by using a high performance liquid chromatograph; 3) carrying out decompressed rotary evaporation and concentration on a 98.5% target peptide solution which is separated and purified, and collecting concentrated liquid for later use; 4) processing the concentrated liquid through a reversed-phase dynamic axial compression column so as to convert the concentrated liquid into acetate by using an ion exchange method; and 5) carrying out decompressed rotary evaporation and concentration and freeze drying on qualified acetate so as to obtain a white flocculent powder product, and inspecting the powder product so that a final product is obtained after qualification. The method for separating and purifying the exenatide provided by the invention is suitable for industrial separation and purification of exenatide.

Description

technical field [0001] The invention relates to a method for large-scale separation and purification of polypeptides, in particular to a method for large-scale separation and purification of Exenatide. Background technique [0002] Diabetes mellitus is a metabolic disorder characterized by chronic hypertension caused by multiple etiologies, mainly caused by defects in insulin secretion or action. Diabetes can be divided into two types, insulin-dependent diabetes (type I diabetes) and non-insulin-dependent diabetes (type II diabetes), of which type II diabetes accounts for more than 90%. According to the statistics of WHO, there are currently 130 million people with type 2 diabetes diagnosed in the world, and my country has more than 40 million people. It is the second largest country with diabetes after India. [0003] Commonly used drugs for treating type II diabetes include: biguanides, sulfonylureas, α-glucosidase inhibitors, thiazolidinediones and insulin. However, stu...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K1/20A61P3/10
Inventor 徐红岩张德志张宏伟杨翼
Owner 滨海吉尔多肽有限公司
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